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Membranes and microtubules of the mitotic apparatus of mammalian cells
JOURNAL
OF ULTRASTRUCTURE
RESEARCH
76, 127-133 (1981)
Membranes and Microtubules of the Mitotic Apparatus of Mammalian Cells NEIDHARD P A W E L E T Z AND E v A - M A R I A F I N Z E
Institute for Cell and Tumorbiology, German Cancer Research Center, D-69 Heidelberg, Federal Republic of Germany Received January 28, 1981 HeLa cells in mitosis are fixed with glutaraldehyde followed by fixation in a saturated aqueous solution of KMn04. In this combination microtubules are preserved and membranes stand out more clearly than after conventional fixation. The microtubular bundles between poles and chromosomes are accompanied by elongated cisternae of the ER, vesicles are found all over the spindle area, within the bundles of microtubules and in the neighborhood of kinetochores. In different regions of the mitotic apparatus microtubules are in close connection with membranes. Some vesicles are in direct contact with elongated cisternae, indicating a close relationship between the two different membranous systems. The role of vesicles as easily transportable "containers" is discussed.
In the last few years evidence on the importance of membranes in the mitotic apparatus has increased (for literature, see Hepler (1977, 1980)). Porter and Machado (1960) described accumulations of membranes at the poles and around the spindle in mitotic cells of higher plants. Harris (1975) found masses of membranous vesicles and cisternae in the mitotic apparatus of fertilized sea urchin eggs. Reports on the ultrastructure of different types of cells in mitosis mentioned membranes in the mitotic apparatus (for literature, see Moll and Paweletz (1980)), but much more attention has been focused on the main constituents of the spindle, the microtubules. Only recently Hepler (1977, 1980) gave a detailed description of membranes and microtubules in plant cells in mitosis. Moll and Paweletz (1980) reported on the membranous systems in and around the mitotic apparatus of mammalian cells after KMnO4 fixation. Since KMnO4 destroys microtubules completely nothing could be said in that paper on the relationship between membranes and microtubules. Paweletz (1978) has studied the membranes in the mitotic apparatus of HeLa cells after conventional fixation with glutaraldehyde and
OsO4. The preservation of microtubules was good; however, that of membranes was relatively poor. Therefore, we developed a method to use a combination of glutaraldehyde and KMnO4 fixation to achieve a good preservation of the microtubules with a good fixation of membranes. Applying this combined fixation to HeLa cells, the relationship between microtubules and membranes in the mitotic apparatus becomes clearer. Recently studies on different cell types show increasing evidence that Ca2+ ions are sequestered by membranes in and around the mitotic apparatus (Schatten and Schatten, 1980; Silver et al., 1980; Wick and Hepler, 1980). There are now strong arguments for the role of membranes in the regulation of the formation of the spindle (Hepler, 1977, 1980). To enhance our knowledge on the control of mitotic events within the cell, detailed studies of the membranous systems in many different cell types during mitosis are necessary. MATERIAL AND METHODS HeLa cells were used for our studies. They were grown on tube slips in monolayer culture in Eagle's minimal essential medium, supplemented with 10% 127 0022-5320/81/080127-07502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
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calf serum, 1% e-glutamine, and 1% nonessential amino acids. The number of cells in mitosis was increased by a thymidine block (2 mM). After careful washing in warm Hanks' balanced salt solution (HBSS) the cells were fixed in 2.5% glutaraldehyde in nutrient medium at ÷37°C for 15 min. This mixutre was prepared immediately before use. The preparations were washed again in HBSS at room temperature and then postfixed in a saturated aqueous solution of KMnO4 at room temperature for 5 min. The length of each of the two fixation periods is the most critical step of the whole procedure. If fixation in glutaraldehyde is too long, the effect of KMnO4 is poor; if KMnO4 is used longer than 5 rain the microtubules are partly or totally destroyed. The procedure given here has proved to be the optimum for HeLa cells under the conditions described above, but it may not be a general procedure for all cell types. After the second fixation the preparations were washed in 25% ethanol until the washing solution remained colorless. Dehydration was carried out in grades of acetone; the embedding occurred in a mixture according to Spurr (1969). Ultrathin sections were cut with a diamond knife on a Reichert Ultramicrotome OmU3, put on formvar-carbon coated grids, stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963), and studied in a Philips 400 electron microscope. RESULTS T h e c e n t r a l p a r t o f a cell in m e t a p h a s e is s h o w n in Fig. 1 at l o w m a g n i f i c a t i o n . T h e t w o p o l e s (P1, P2) o f t h e s p i n d l e a r e c h a r a c t e r i z e d b y an a c c u m u l a t i o n o f m e m b r a n o u s s t r u c t u r e s . A t P2 a s t a r - l i k e a r r a n g e ment of membranes becomes visible. Within the mitotic apparatus accumulations o f m e m b r a n e s (M) a n d b u n d l e s o f m i c r o t u b u l e s (Mt) a r e p r e s e n t . A l l large p a r t i c l e s except the chromosomes remain excluded from the mitotic apparatus. While particles l a r g e r t h a n r i b o s o m e s , w h i c h at h i g h e r m a g nification (Figs. 3 - 6 ) a p p e a r as m e m b r a n o u s v e s i c l e s , a r e d i s t r i b u t e d all o v e r t h e spindle area, the elongated cisternae appear
at the p e r i p h e r y o f t h e m i t o t i c a p p a r a t u s a n d a r e c o n c e n t r a t e d in t h e r e g i o n a r o u n d t h e (virtual) l o n g axis o f the s p i n d l e (Fig. 2). T h e y m o s t l y r u n p a r a l l e l to t h e m i c r o tubular bundles. Figure 2 depicts part of the c e n t r a l r e g i o n o f t h e spindle. V e s i c l e s , cisternae, and microtubules are intermingled with each other; therefore direct contacts between microtubules and membranes are h i g h l y p r o b a b l e . S o m e v e s i c l e s a r e in d i r e c t c o n n e c t i o n w i t h c i s t e r n a e (Fig. 3), s u g g e s t ing t h a t t h e s e a r e p r o d u c t s o f a f u s i o n o r a pinching-off process. Cisternae within the domain of the spindle are mostly accumul a t e d in t h e c e n t e r o f the s p i n d l e w i t h mic r o t u b u l e b u n d l e s o n b o t h sides ( F i g s . 1, 2). T a n g e n t i a l s e c t i o n s t h r o u g h t h e c i s t e r n a e (Fig. 2) r e v e a l a c o n f l u e n t s y s t e m o f lacunae and bleb-like dilatations of the membranous sacs. Longitudinal or transverse sections of the membranes only demonstrate elongated flattened cisternae. The p e r i p h e r a l s h e a t h o f m e m b r a n e s (Fig. 2, pM) consists of overlapping cisternae. M o s t o f the m e m b r a n e profiles w i t h i n t h e m i t o t i c a p p a r a t u s e x t e n d f r o m t h e p o l a r reg i o n to t h e c h r o m o s o m e s (Fig. 2), a n d s o m e e v e n p e n e t r a t e o r c r o s s the m e t a p h a s e p l a t e (Fig. 4), t h u s b e c o m i n g c o n n e c t e d to t h e membranous system of the other half-spindle. In more detailed studies of the metaphase p l a t e o n e c a n see t h a t m e m b r a n e s a r e c o n nected to microtubules directly or via b r i d g e s ( F i g s . 3, 5, 6, a r r o w s ) ; d i r e c t c o n tact between membranes and chromosomes c a n b e f o u n d in s o m e r e g i o n s (Fig. 4). V e r y short cisternae and mostly vesicles are p r e s e n t in a r e g i o n m o r e d i s t a n t f r o m t h e z o n e a r o u n d t h e l o n g axis. S i n c e s m a l l b u n dles o f m i c r o t u b u l e s p a s s b y t h e c h r o m o -
FIG. 1. Survey of the central part of a cell in metaphase-early anaphase. The mitotic apparatus is surrounded by membranous cisternae. Two poles (P1, P2) are visible; at P2 a star-like arrangement of membranes can be found. Bundles of microtubules (Mt) and accumulations of membranes (M) are seen within the mitotic apparatus. × 9600. FIG. 2. Part of the mitotic apparatus. The membranes (M) around the long axis of the spindle are inflated and accompanied by bundles of microtubules (Mt). The membranes extend from the polar region (P) to the chromosomes (Ch). The mitotic apparatus is surrounded by overlapping pieces of ER cisternae (pM). x 21 000.
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FIG. 3. An accumulation of membranes (M) among bundles of microtubules (Mt). Some vesicles are in close connection with elongated cisternae (arrowheads). They represent either fusion or pinched-off products. An elongated cisterna is in direct contact with a microtubule (arrow). × 42 000.
131
MEMBRANES AND MICROTUBULES IN MITOSIS
somes, the cisternae near the chromosomes, in addition to interacting with the chromosomes, are also closely associated with microtubules (Fig. 4). Near the kinetochores tube-like membranes can sometimes be found. These tubes are somewhat distant from the kinetochore microtubules. Within the bundles and in their direct neighborhood, vesicles are prominent. Some of them are connected with microtubules directly or via bridges (Fig. 6, arrows). Cisternae of the ER type could not be seen at or in the vicinity of kinetochores, and unlike the vesicles, they are not found within microtubule bundles. A slight accumulation of vesicles near the kinetochores could be observed. During anaphase the cisternae, which in metaphase run from the poles to the chromosomes, accumulate in front of the moving chromosomes and do not enter the midbody region. Vesicles already known from all parts of the former metaphase spindle enter the midbody obviously without any hindrance. They are spread all over the midbody. At higher magnification the vesicles become prominent; some are located in the vicinity of microtubules (Fig. 5, arrows), and some others are seen within the microtubule bundles itself. In addition, the tube-like membranes, which were already found during metaphase in the neighborhood of the kinetochores, are also present within the midbody region, near the microtubule bundles but not within them (Fig. 5). Figure 7 is a scheme summarizing the different types of membranes visualized by the present fixation technique. This drawing indicates a possible relationship between intranuclear spindles and polar fenestrae in lower organisms and the HeLa mitotic apparatus.
o
oq-rron
~/'77g
FIG. 7. A schematic drawing depicts the different types of membranes in and around the mitotic apparatus.
DISCUSSION
The use of KMnO4 as fixative for the membranes in the mitotic apparatus of mammalian cells (Moll and Paweletz, 1980) has revealed new insights into the membranous system of cells in mitosis. These data are, however, incomplete since a relationship between membranes and microtubules cannot be studied after KMnO4 fixation. Conventional fixation (glutaraldehyde and osmium tetroxide) does not preserve the membranes sufficiently well to obtain a survey on both membranes and microtubules (Paweletz, 1980). The sequential use of first glutaraldehyde and second KMn04 as fixatives for the same cell has revealed the structural relations between spindle elements (microtubules, chromosomes, kinetochores) and membranes. Due to the
FIG. 4. Membranes (M) accompany bundles of microtubules (Mt) and chromosomes. × 31 500. Fro. 5. Part of the midbody. Microtubules (Mt) are in close connection with tube-like membranous cisternae (arrows). x 44 000. Fro. 6. Vesicles can be found within bundles of kinetochore microtubules (kMt). They are connected by bridges to single microtubules (arrows). A kinetochore (K) is visible at the left side. x 66 000.
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membranous sheath around the mitotic apparatus, microtubules and chromosomes can come into contact with membranes and with compounds which are sequestered in them. In addition, the cisternae of the ER running either from pole to pole or from pole to the metaphase plate, increase these areas of contact. The data presented here are in full agreement with the data obtained for plant cells by Hepler (1980). The accumulation of cisternae in the polar regions in plants coincides with the astral arrangement of membranes at the poles of mammalian cells. This different arrangement seen in relation to the different forms of the spindle in the two cell types (astral in mammalian cells, anastral in plant cells) indicates clearly an interrelationship between microtubules and membranes in the polar regions. Both systems obviously support each other, giving rise to microtubular asters observed in preparations when membranes are not well preserved ~s well as to membranous ones observed in preparations when microtubules are not preserved at all. From this arrangement, however, it cannot be decided if the membranous system orients the microtubular system or vice versa. It could be further supposed that both systems interact to stabilize each other (see also Hepler, 1980). Hepler (1977, 1980) has discussed the role of membranes in the mitotic apparatus of plant cells in detail. Since it can be assumed that there are only minor differences in this role between plant and mammalian cells, this aspect will not be discussed here. It is, however, necessary to discuss whether or not the arrangement of the membranous system can fit the purpose, namely, to create a Ca~+-sequestering system within the spindle, to generate discrete concentrations in different parts of the mitotic apparatus. If there is a diffusion of regulating molecules or ions from the interior of the cisternae and if these substances are involved in the formation of the spindle, are
there enough membranes to enable all parts of the spindle to be regulated? The peripheral bundles of microtubules are very close to the peripheral membranous sheath. The central bundles, however, need additional membranes in the center. This is obviously realized by the penetration of membranes from the poles toward the metaphase plate around the (virtual) long axis of the spindle. Though there is not yet direct evidence, it can be supposed that these membranes are in connection with cisternae of the membranous aster at the poles and the membranous sheath at the periphery. Thus, a continuous system of channels can be formed to assure an uninhibited, rapid transport of regulating substances from one part of the spindle to another, e.g., from the poles to the chromosomes or vice versa (see also Hepler, 1980). In addition to this channel system which seems to be relatively rigid a more mobile "shuttle" system must be postulated which enables the cell to transport '~containers" all across the mitotic apparatus. The number of °'containers" can influence the concentrations of molecules or ions necessary for the regulation of the formation of the spindle. Such " c o n tainers" are found within the domain of the mitotic apparatus in the form of membranous vesicles. Though these vesicles can be found all over the spindle, a slight accumulation is seen near the poles in early stages (Moll and Paweletz, 1980) and later on in the neighborhood of the kinetochores. In addition, these "containers" are small enough to penetrate even the bundles of MTs. In some cases we have found vesicles in direct connection with elongated cisternae. It cannot yet be decided whether these are products of a fusion or a pinching-off process. Nevertheless a direct interrelationship between "channels" and "containers" can be demonstrated. Besides the star-like arrangement of elongated cisternae in the polar region of animal cells only minor differences exist in the arrangement of cisternae between plant
MEMBRANES AND MICROTUBULES IN MITOSIS
and animal cells (Hepler, 1980; Moll and Paweletz, 1980). Larger differences, however, seem to exist in the existence of vesicles. Using a special fixation procedure for membranes of the ER in barley cells, Hepler (1980) could not demonstrate vesicles. After conventional fixation he showed a few of them. However, Porter and Machado (1980) observed, in KMnO4-fixed cells of the onion root tip, that large amounts of vesicles exist within the domain of the mitotic apparatus. Therefore, it can be argued that vesicles are indeed present in barley cells and the differences between Hepler's (1980) and our studies are due to different preparation techniques. The question arises for plant (Hepler, 1980) as well as for mammalian material, whether the accumulation of membranes around the mitotic apparatus is only due to the change in shape by rounding up of the cell bringing more membranes from the periphery to the center or to the formation of new cisternae. Observations of the ultrastructure of HeLa cells in mitosis after the application of antimitotic drugs (George et al., 1965) and recent data on rounded-up interphase cells indicate that the increase in membranes around the mitotic apparatus
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is, at least in large part, due to the production of new membranous cisternae. We thank Mrs. L. D6ring and Miss I. Purkert for technical assistance and Dr. R. Tchao for critical reading of the manuscript.
REFERENCES GEORGE, L., JOURNEY, L. J., AND GOLDSTEIN, M. N. (1965) J. Nat. Cancer Inst. 35, 355-375. HARRIS, P. (1975) Exp. Cell Res. 94, 409-425. HEPLER, P. K. (1977) in ROST, T. L., AND GIFFORD, E. M. (Eds.), Mechanism and Control of Cell Division, pp. 212-232, Dowden, Hutchinson, & Ross, Stroudsburg, Pa. HEPLER, P. K. (1980) J. Cell Biol. 86, 490-499. MOLL, E., AND PAWELETZ, N. (1980) Eur. J. Cell Biol. 21,280-287. PAWELETZ, N. (1978) in DIRKSEN, E. R., PRESCOTT, D., AND FOX, C. F. (Eds.), Cell Reproduction: Honoring Daniel Mazia, pp. 515-523, Academic Press, New York. PORTER, K. R., AND MACHAOO, R. (1960) d. Biophys. Biochem. Cytol. 7, 167-180. REYNOLDS, E. S. (1963) J. CellBiol. 17, 208-212. SCHATTEN, G., AND SCHATTEN, H. (1980) Eur. J. Cell Biol. 22, 314. SILVER, R. B., COLE, R. D., AND CANDE, W. Z. (1980) Cell 19, 505-516. SPURR, A. R. (1969) J. Ultrastruct. Res. 26, 31-43. WATSON, M. L. (1958) J. Biophys. Biochern. Cytol. 4, 475-478. WICK, S. M., AND HEPLER, P. K. (1980) J. CellBiol. 86, 500-513.
OF ULTRASTRUCTURE
RESEARCH
76, 127-133 (1981)
Membranes and Microtubules of the Mitotic Apparatus of Mammalian Cells NEIDHARD P A W E L E T Z AND E v A - M A R I A F I N Z E
Institute for Cell and Tumorbiology, German Cancer Research Center, D-69 Heidelberg, Federal Republic of Germany Received January 28, 1981 HeLa cells in mitosis are fixed with glutaraldehyde followed by fixation in a saturated aqueous solution of KMn04. In this combination microtubules are preserved and membranes stand out more clearly than after conventional fixation. The microtubular bundles between poles and chromosomes are accompanied by elongated cisternae of the ER, vesicles are found all over the spindle area, within the bundles of microtubules and in the neighborhood of kinetochores. In different regions of the mitotic apparatus microtubules are in close connection with membranes. Some vesicles are in direct contact with elongated cisternae, indicating a close relationship between the two different membranous systems. The role of vesicles as easily transportable "containers" is discussed.
In the last few years evidence on the importance of membranes in the mitotic apparatus has increased (for literature, see Hepler (1977, 1980)). Porter and Machado (1960) described accumulations of membranes at the poles and around the spindle in mitotic cells of higher plants. Harris (1975) found masses of membranous vesicles and cisternae in the mitotic apparatus of fertilized sea urchin eggs. Reports on the ultrastructure of different types of cells in mitosis mentioned membranes in the mitotic apparatus (for literature, see Moll and Paweletz (1980)), but much more attention has been focused on the main constituents of the spindle, the microtubules. Only recently Hepler (1977, 1980) gave a detailed description of membranes and microtubules in plant cells in mitosis. Moll and Paweletz (1980) reported on the membranous systems in and around the mitotic apparatus of mammalian cells after KMnO4 fixation. Since KMnO4 destroys microtubules completely nothing could be said in that paper on the relationship between membranes and microtubules. Paweletz (1978) has studied the membranes in the mitotic apparatus of HeLa cells after conventional fixation with glutaraldehyde and
OsO4. The preservation of microtubules was good; however, that of membranes was relatively poor. Therefore, we developed a method to use a combination of glutaraldehyde and KMnO4 fixation to achieve a good preservation of the microtubules with a good fixation of membranes. Applying this combined fixation to HeLa cells, the relationship between microtubules and membranes in the mitotic apparatus becomes clearer. Recently studies on different cell types show increasing evidence that Ca2+ ions are sequestered by membranes in and around the mitotic apparatus (Schatten and Schatten, 1980; Silver et al., 1980; Wick and Hepler, 1980). There are now strong arguments for the role of membranes in the regulation of the formation of the spindle (Hepler, 1977, 1980). To enhance our knowledge on the control of mitotic events within the cell, detailed studies of the membranous systems in many different cell types during mitosis are necessary. MATERIAL AND METHODS HeLa cells were used for our studies. They were grown on tube slips in monolayer culture in Eagle's minimal essential medium, supplemented with 10% 127 0022-5320/81/080127-07502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
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calf serum, 1% e-glutamine, and 1% nonessential amino acids. The number of cells in mitosis was increased by a thymidine block (2 mM). After careful washing in warm Hanks' balanced salt solution (HBSS) the cells were fixed in 2.5% glutaraldehyde in nutrient medium at ÷37°C for 15 min. This mixutre was prepared immediately before use. The preparations were washed again in HBSS at room temperature and then postfixed in a saturated aqueous solution of KMnO4 at room temperature for 5 min. The length of each of the two fixation periods is the most critical step of the whole procedure. If fixation in glutaraldehyde is too long, the effect of KMnO4 is poor; if KMnO4 is used longer than 5 rain the microtubules are partly or totally destroyed. The procedure given here has proved to be the optimum for HeLa cells under the conditions described above, but it may not be a general procedure for all cell types. After the second fixation the preparations were washed in 25% ethanol until the washing solution remained colorless. Dehydration was carried out in grades of acetone; the embedding occurred in a mixture according to Spurr (1969). Ultrathin sections were cut with a diamond knife on a Reichert Ultramicrotome OmU3, put on formvar-carbon coated grids, stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963), and studied in a Philips 400 electron microscope. RESULTS T h e c e n t r a l p a r t o f a cell in m e t a p h a s e is s h o w n in Fig. 1 at l o w m a g n i f i c a t i o n . T h e t w o p o l e s (P1, P2) o f t h e s p i n d l e a r e c h a r a c t e r i z e d b y an a c c u m u l a t i o n o f m e m b r a n o u s s t r u c t u r e s . A t P2 a s t a r - l i k e a r r a n g e ment of membranes becomes visible. Within the mitotic apparatus accumulations o f m e m b r a n e s (M) a n d b u n d l e s o f m i c r o t u b u l e s (Mt) a r e p r e s e n t . A l l large p a r t i c l e s except the chromosomes remain excluded from the mitotic apparatus. While particles l a r g e r t h a n r i b o s o m e s , w h i c h at h i g h e r m a g nification (Figs. 3 - 6 ) a p p e a r as m e m b r a n o u s v e s i c l e s , a r e d i s t r i b u t e d all o v e r t h e spindle area, the elongated cisternae appear
at the p e r i p h e r y o f t h e m i t o t i c a p p a r a t u s a n d a r e c o n c e n t r a t e d in t h e r e g i o n a r o u n d t h e (virtual) l o n g axis o f the s p i n d l e (Fig. 2). T h e y m o s t l y r u n p a r a l l e l to t h e m i c r o tubular bundles. Figure 2 depicts part of the c e n t r a l r e g i o n o f t h e spindle. V e s i c l e s , cisternae, and microtubules are intermingled with each other; therefore direct contacts between microtubules and membranes are h i g h l y p r o b a b l e . S o m e v e s i c l e s a r e in d i r e c t c o n n e c t i o n w i t h c i s t e r n a e (Fig. 3), s u g g e s t ing t h a t t h e s e a r e p r o d u c t s o f a f u s i o n o r a pinching-off process. Cisternae within the domain of the spindle are mostly accumul a t e d in t h e c e n t e r o f the s p i n d l e w i t h mic r o t u b u l e b u n d l e s o n b o t h sides ( F i g s . 1, 2). T a n g e n t i a l s e c t i o n s t h r o u g h t h e c i s t e r n a e (Fig. 2) r e v e a l a c o n f l u e n t s y s t e m o f lacunae and bleb-like dilatations of the membranous sacs. Longitudinal or transverse sections of the membranes only demonstrate elongated flattened cisternae. The p e r i p h e r a l s h e a t h o f m e m b r a n e s (Fig. 2, pM) consists of overlapping cisternae. M o s t o f the m e m b r a n e profiles w i t h i n t h e m i t o t i c a p p a r a t u s e x t e n d f r o m t h e p o l a r reg i o n to t h e c h r o m o s o m e s (Fig. 2), a n d s o m e e v e n p e n e t r a t e o r c r o s s the m e t a p h a s e p l a t e (Fig. 4), t h u s b e c o m i n g c o n n e c t e d to t h e membranous system of the other half-spindle. In more detailed studies of the metaphase p l a t e o n e c a n see t h a t m e m b r a n e s a r e c o n nected to microtubules directly or via b r i d g e s ( F i g s . 3, 5, 6, a r r o w s ) ; d i r e c t c o n tact between membranes and chromosomes c a n b e f o u n d in s o m e r e g i o n s (Fig. 4). V e r y short cisternae and mostly vesicles are p r e s e n t in a r e g i o n m o r e d i s t a n t f r o m t h e z o n e a r o u n d t h e l o n g axis. S i n c e s m a l l b u n dles o f m i c r o t u b u l e s p a s s b y t h e c h r o m o -
FIG. 1. Survey of the central part of a cell in metaphase-early anaphase. The mitotic apparatus is surrounded by membranous cisternae. Two poles (P1, P2) are visible; at P2 a star-like arrangement of membranes can be found. Bundles of microtubules (Mt) and accumulations of membranes (M) are seen within the mitotic apparatus. × 9600. FIG. 2. Part of the mitotic apparatus. The membranes (M) around the long axis of the spindle are inflated and accompanied by bundles of microtubules (Mt). The membranes extend from the polar region (P) to the chromosomes (Ch). The mitotic apparatus is surrounded by overlapping pieces of ER cisternae (pM). x 21 000.
MEMBRANES AND M I C R O T U B U L E S IN MITOSIS
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FIG. 3. An accumulation of membranes (M) among bundles of microtubules (Mt). Some vesicles are in close connection with elongated cisternae (arrowheads). They represent either fusion or pinched-off products. An elongated cisterna is in direct contact with a microtubule (arrow). × 42 000.
131
MEMBRANES AND MICROTUBULES IN MITOSIS
somes, the cisternae near the chromosomes, in addition to interacting with the chromosomes, are also closely associated with microtubules (Fig. 4). Near the kinetochores tube-like membranes can sometimes be found. These tubes are somewhat distant from the kinetochore microtubules. Within the bundles and in their direct neighborhood, vesicles are prominent. Some of them are connected with microtubules directly or via bridges (Fig. 6, arrows). Cisternae of the ER type could not be seen at or in the vicinity of kinetochores, and unlike the vesicles, they are not found within microtubule bundles. A slight accumulation of vesicles near the kinetochores could be observed. During anaphase the cisternae, which in metaphase run from the poles to the chromosomes, accumulate in front of the moving chromosomes and do not enter the midbody region. Vesicles already known from all parts of the former metaphase spindle enter the midbody obviously without any hindrance. They are spread all over the midbody. At higher magnification the vesicles become prominent; some are located in the vicinity of microtubules (Fig. 5, arrows), and some others are seen within the microtubule bundles itself. In addition, the tube-like membranes, which were already found during metaphase in the neighborhood of the kinetochores, are also present within the midbody region, near the microtubule bundles but not within them (Fig. 5). Figure 7 is a scheme summarizing the different types of membranes visualized by the present fixation technique. This drawing indicates a possible relationship between intranuclear spindles and polar fenestrae in lower organisms and the HeLa mitotic apparatus.
o
oq-rron
~/'77g
FIG. 7. A schematic drawing depicts the different types of membranes in and around the mitotic apparatus.
DISCUSSION
The use of KMnO4 as fixative for the membranes in the mitotic apparatus of mammalian cells (Moll and Paweletz, 1980) has revealed new insights into the membranous system of cells in mitosis. These data are, however, incomplete since a relationship between membranes and microtubules cannot be studied after KMnO4 fixation. Conventional fixation (glutaraldehyde and osmium tetroxide) does not preserve the membranes sufficiently well to obtain a survey on both membranes and microtubules (Paweletz, 1980). The sequential use of first glutaraldehyde and second KMn04 as fixatives for the same cell has revealed the structural relations between spindle elements (microtubules, chromosomes, kinetochores) and membranes. Due to the
FIG. 4. Membranes (M) accompany bundles of microtubules (Mt) and chromosomes. × 31 500. Fro. 5. Part of the midbody. Microtubules (Mt) are in close connection with tube-like membranous cisternae (arrows). x 44 000. Fro. 6. Vesicles can be found within bundles of kinetochore microtubules (kMt). They are connected by bridges to single microtubules (arrows). A kinetochore (K) is visible at the left side. x 66 000.
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PAWELETZ AND FINZE
membranous sheath around the mitotic apparatus, microtubules and chromosomes can come into contact with membranes and with compounds which are sequestered in them. In addition, the cisternae of the ER running either from pole to pole or from pole to the metaphase plate, increase these areas of contact. The data presented here are in full agreement with the data obtained for plant cells by Hepler (1980). The accumulation of cisternae in the polar regions in plants coincides with the astral arrangement of membranes at the poles of mammalian cells. This different arrangement seen in relation to the different forms of the spindle in the two cell types (astral in mammalian cells, anastral in plant cells) indicates clearly an interrelationship between microtubules and membranes in the polar regions. Both systems obviously support each other, giving rise to microtubular asters observed in preparations when membranes are not well preserved ~s well as to membranous ones observed in preparations when microtubules are not preserved at all. From this arrangement, however, it cannot be decided if the membranous system orients the microtubular system or vice versa. It could be further supposed that both systems interact to stabilize each other (see also Hepler, 1980). Hepler (1977, 1980) has discussed the role of membranes in the mitotic apparatus of plant cells in detail. Since it can be assumed that there are only minor differences in this role between plant and mammalian cells, this aspect will not be discussed here. It is, however, necessary to discuss whether or not the arrangement of the membranous system can fit the purpose, namely, to create a Ca~+-sequestering system within the spindle, to generate discrete concentrations in different parts of the mitotic apparatus. If there is a diffusion of regulating molecules or ions from the interior of the cisternae and if these substances are involved in the formation of the spindle, are
there enough membranes to enable all parts of the spindle to be regulated? The peripheral bundles of microtubules are very close to the peripheral membranous sheath. The central bundles, however, need additional membranes in the center. This is obviously realized by the penetration of membranes from the poles toward the metaphase plate around the (virtual) long axis of the spindle. Though there is not yet direct evidence, it can be supposed that these membranes are in connection with cisternae of the membranous aster at the poles and the membranous sheath at the periphery. Thus, a continuous system of channels can be formed to assure an uninhibited, rapid transport of regulating substances from one part of the spindle to another, e.g., from the poles to the chromosomes or vice versa (see also Hepler, 1980). In addition to this channel system which seems to be relatively rigid a more mobile "shuttle" system must be postulated which enables the cell to transport '~containers" all across the mitotic apparatus. The number of °'containers" can influence the concentrations of molecules or ions necessary for the regulation of the formation of the spindle. Such " c o n tainers" are found within the domain of the mitotic apparatus in the form of membranous vesicles. Though these vesicles can be found all over the spindle, a slight accumulation is seen near the poles in early stages (Moll and Paweletz, 1980) and later on in the neighborhood of the kinetochores. In addition, these "containers" are small enough to penetrate even the bundles of MTs. In some cases we have found vesicles in direct connection with elongated cisternae. It cannot yet be decided whether these are products of a fusion or a pinching-off process. Nevertheless a direct interrelationship between "channels" and "containers" can be demonstrated. Besides the star-like arrangement of elongated cisternae in the polar region of animal cells only minor differences exist in the arrangement of cisternae between plant
MEMBRANES AND MICROTUBULES IN MITOSIS
and animal cells (Hepler, 1980; Moll and Paweletz, 1980). Larger differences, however, seem to exist in the existence of vesicles. Using a special fixation procedure for membranes of the ER in barley cells, Hepler (1980) could not demonstrate vesicles. After conventional fixation he showed a few of them. However, Porter and Machado (1980) observed, in KMnO4-fixed cells of the onion root tip, that large amounts of vesicles exist within the domain of the mitotic apparatus. Therefore, it can be argued that vesicles are indeed present in barley cells and the differences between Hepler's (1980) and our studies are due to different preparation techniques. The question arises for plant (Hepler, 1980) as well as for mammalian material, whether the accumulation of membranes around the mitotic apparatus is only due to the change in shape by rounding up of the cell bringing more membranes from the periphery to the center or to the formation of new cisternae. Observations of the ultrastructure of HeLa cells in mitosis after the application of antimitotic drugs (George et al., 1965) and recent data on rounded-up interphase cells indicate that the increase in membranes around the mitotic apparatus
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is, at least in large part, due to the production of new membranous cisternae. We thank Mrs. L. D6ring and Miss I. Purkert for technical assistance and Dr. R. Tchao for critical reading of the manuscript.
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